[Cytometry] Forward Scatter Issues with BD CantoII
g.nebe-von-caron at alere.com
Wed Apr 17 13:23:42 EDT 2013
Looks like you are almost on top of it with cleaning your system and it would be nice to get some data to look at when triggering at various noise level signals, e.g. SSC and FL1 height at 200 with the voltage settings raised until further increase caused a rapid increase in event rate when running a clean sheath system and 100nm filtered sheath as a sample. This should give you photon noise in the hope that the crap in the sheath end is limited and most instruments are not too bad on this when using side scatter and fluorescence triggering
When measuring bacteria back in the eighties it was already recognised that one of the critical things was the cleanliness of the optical path including the flow cell, the beam shaper, the mirrors and cleaning the Brewster window of the laser to ensure the beam was in Tem00 mode. However the use of forward scatter in conventional instruments is not recommended for small particle detection unless you are in full control of your optics. As you may be well aware, the forward scatter of your instrument does not bare any straight forward relationship to particle size due to the huge impact of small angular variations in the scatter geometry. Kevin Beckers presentation in San Diego in 2002 showed the lack of monotonous forward scatter in a poster of which I still have an elusive copy and referenced on the list https://lists.purdue.edu/pipermail/cytometry/2007-March/032612.html. Unfortunately the link is not active any more but you find the data also in Howard's Practical Flow cytometry in the light scatter section which I would urge everyone to read anyhow. For small particles you gave far less of that problem but also so far less signal so they drown in the noise of the scattered photons in the projection path anyhow.
I showed the problems of comparing beads and biological particles in http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20696/pdf and in
Nebe-von Caron, G., Badley, R.A., 1996. Bacterial characterization by flow cytometry. In: Al-Rubeai, M., Emery, A.N. (Eds.)
Flow Cytometry Applications in Cell Culture. Marcel Dekker, New York, pp. 257-290, where the 2230nm beads show less signal than the 1160nm beads in my modified forward scatter equal signal in the standard forward scatter of the Elite but higher signal in the side scatter.
Now I still try to be in San Diego with the informal help of Kevin as I am unable to come to the US. Is not that they won't let me in as some of you might suggest but because of my "membrane integrity problem" But don't worry I am the living prove that a lack of membrane integrity does not always indicate the absence of viability. I hope Kevin can either put me on a screen at his booth or in some convenient location for a virtual "bring a beer workshop" to talk about small particle trouble in the light of scatter.
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Bryant Hanks
Sent: 16 April 2013 20:54
To: Cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Forward Scatter Issues with BD CantoII
Thanks to all who responded to my inquiry.
I focused mostly on the fluidic pathway and was reminded to check the optical pathway for cleanliness also.
Per BD FSE Craig Hayes, I carefully cleaned the prisms and lenses with ETOH and lens paper. With a lint free Q-tip and hemostats, I also gently wiped the sides of the flowcell itself. This yielded a huge improvement in the 1.00 um bead FSC-A histogram.
Another suggestion to decrease the Window Extension, (under the Laser tab), from 7 to 2 was very helpful.
Again, thanks to all who contribute to this website and make it such a treasure of information.
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